Methylcobalamin is a coenzyme-type vitamin B12 existing in blood and cerebrospinal fluid and is excellent in migrating ability to nervous tissues as compared with other B12 homologs. Biochemically, it exhibits a pharmacological action of accelerating metabolism of nucleic acids, proteins and lipids by methyl group rearrangement and thereby restoring damaged nervous tissues. Based on these properties, it has been clinically employed for preventing, treating or improving peripheral neuropathy such as diabetic neuropathy and polyneuritis, particularly numbness, pain and paralysis, and is also effective in megaloblastic anemia owing to vitamin B12 deficiency, and thus, it is an important vitamin.
Accordingly, the present invention relates to an industrially excellent and novel process for producing methylcobalamin useful as medicines.
Methylcobalamin has been hitherto produced mainly by the following synthetic methods:
(1) a method of reacting hydroxocobalamin with a dicarboxylic acid monomethyl ester in the presence of a metal powder (JP-A 49-47899);
(2) a method of reacting cyanocobalamin with monomethyl oxalate in the presence of a metal powder in hydrous methanol (JP-A 50-41900);
(3) a method of reacting hydroxocobalamin with methylmercury iodide or ammonium methylhexafluorosilicate (JP-B 50-38120); and
(4) a method of reacting cyanocobalamin with methyl iodide in the presence of sodium borohydride (JP-B 45-38059).
However, dicarboxylic acid monomethyl esters such as monomethyl oxalate to be used in the methods (1) and (2) are not commercially available and hence are necessary to prepare in use, so that it is impossible to utilize them industrially. Furthermore, zinc powder to be used as the metal powder is a heavy metal and hence it is inevitable to take measures for preventing its contamination into products and for protecting the environment, so that the powder is industrially not preferable.
Moreover, methylmercury iodide to be used in (3) is a pollutant and hence cannot be employed industrially. Furthermore, ammonium methylhexafluorosilicate is also not commercially available and hence is necessary to prepare in use, so that it is impossible to utilize it industrially.
On the other hand, the synthetic method (4) is a very excellent method in view of yield and product purity, but is not satisfactory as an industrial process because methyl iodide has an extremely low boiling point (41 to 43xc2x0 C.) and is difficult to handle. Furthermore, from the viewpoint of protecting working environment or natural environment, the use of methyl iodide assigned as a specified chemical substance and having toxicity such as possibility of carcinogenicity is by no means preferable in view of industrial health of factory workers. Moreover, in order to obtain highly pure methylcobalamin by the method of using methyl iodide, operation for purification by one or more kinds of column chromatography is usually necessary, which is a serious problem from operational viewpoint and viewpoint of production cost. In addition, the quantity of organic solvents for use in the column purification is large and also waste liquid quantity tends to be enormous.
Thus, an industrially excellent process for producing methylcobalamin is not completely established yet and hence a novel excellent method has been desired.
The present inventors have extensively studied for the purpose of improving the above problems. As a result, surprisingly, they have found that aimed methylcobalamin can be conveniently, safely, and inexpensively obtained in high yields by the below-mentioned method, and thus accomplished the present invention.
Accordingly, the present invention provides an industrially excellent process for producing methylcobalamin, particularly a novel process using no methyl iodide and no purification by column chromatography.
The following will explain the present invention in detail.
The present invention relates to a process for producing methylcobalamin (V), which is represented by the following chemical reaction formula:
Cobalamin-CN or Cobalamin-OHxe2x86x92Cobalamin-CH3 
Cyanocobalamin (I), hydroxocobalamin (II), and methylcobalamin (V) according to the present invention are known natural compounds and are represented by the following chemical formula:
Cyanocobalamin, CAS Res. No.: 68-19-9
Hydroxocobalamin, CAS Res. No.: 13422-51-0
Methylcobalamin, CAS Res. No.: 13422-55-4 
R2xe2x95x90CN: Cyanocobalamin (I)
R2xe2x95x90OH: Hydroxocobalamin (II)
R2xe2x95x90CH3: Methylcobalamin (V)
The characteristic feature of the present invention is that a highly pure methylcobalamin equal to or superior to the product purified by column chromatography can be conveniently obtained in high yields only by methylating cyanocobalamin (I) or hydroxocobalamin (II) in the presence of a reducing agent (III) and a water-soluble methylating agent (IV) usually in an aqueous solution or a hydrous organic solvent, if necessary, precipitating the reaction product which is hardly soluble in water as crystals or precipitates, and then separating and treating it.
The water-soluble methylating agent (IV) in the present invention is not limited as far as it""s solubility in water is 2% or more, and specifically includes trimethylsulfur derivatives (VI) represented by the following formula, for example. 
wherein X represents a halogen atom or methoxysulfonyloxy group; and n represents 0 or 1.
Examples of the trimethylsulfur derivatives (VI) include the following compounds but they are not limited thereto.
(1) Trimethylsulfoxonium iodide, CAS Res. No.: 1774-47-6
(2) Trimethylsulfonium iodide, CAS Res. No.: 2181-42-2
(3) Trimethylsulfoxonium chloride, CAS Res. No.: 5034-06-0
(4) Trimethylsulfonium chloride, CAS Res. No.: 3086-29-1
(5) Trimethylsulfoxonium bromide, CAS Res. No.: 3084-53-5
(6) Trimethylsulfoxonium bromide, CAS Res. No.: 25596-24-1
(7) Trimethylsulfonium methyl sulfate, CAS Res. No.: 2181-44-4
All these compounds are known products and, in particular, trimethylsulfoxonium iodide, trimethylsulfonium iodide, trimethylsulfoxonium chloride, trimethylsulfoxonium bromide and trimethylsulfonium bromide are inexpensive and available as industrial starting materials. Moreover, trimethylsulfonium chloride can be easily synthesized and available by the method described in Tetrahedron Lett., 27, 1233 (1986) (B. Byrne et al.).
Among the trimethylsulfur derivatives (VI), trimethylsulfoxonium bromide, trimethylsulfonium bromide, trimethylsulfoxonium chloride and trimethylsulfonium chloride particularly exhibit a high solubility in water and have a characteristic that the use in a smaller amount affords highly pure methylcobalamin in high yields.
The amount of the trimethylsulfur derivative (VI) to be used is not particularly limited, but it is used in an amount of usually 1.0 to 5 equivalents, preferably 1.1 to 4.5 equivalents and more preferably 1.2 to 4 equivalents to cyanocobalamin (I) or hydroxocobalamin (II).
The reducing agent (III) according to the present invention is not particularly limited as far as it is a reducing agent employable in the synthesis of cyanocobalamin (I) or hydroxocobalamin (II). More specifically, examples thereof include sodium borohydride.
The amount of the reducing agent (III) to be used is not particularly limited, but it is used in an amount of usually 5 to 30 equivalents, preferably 8 to 25 equivalents and more preferably 10 to 20 equivalents to cyanocobalamin (I) or hydroxocobalamin (II).
The process according to the present invention enables the production of highly pure methylcobalamin in high yields using no metal ion or using only a small amount thereof as a cyan ion-trapping agent, and the process exhibits an extremely excellent effect in view that no problem arises at removal of metal ion products, which is difficult to filter, from the system.
Generally, when and methyl iodide is used as a methylating agent, ferrous sulfate is used as a cyan ion-trapping agent in combination with those agents in most cases, and it is necessary to use ferrous sulfate in an amount of at least 30% by weight or more relative to cyanocobalamin (I) or hydroxocobalamin (II).
However, in the present invention, it is possible to obtain highly pure methylcobalamin in high yields because methylation proceeds even when no ferrous sulfate is used as a cyan ion-trapping agent.
Furthermore, in the case that ferrous sulfate is used in a small amount as a cyan ion-trapping agent, the reaction proceeds more rapidly and highly pure methylcobalamin can be obtained in high yields by the same post-treatment as in the case that no ferrous sulfate is used. Moreover, in the case that cobalt chloride is used in a small amount, highly pure methylcobalamin can be also obtained in high yields because the methylation proceeds highly selectively and hence the production of impurities is inhibited.
Therefore, the present invention also relates to a process for producing methylcobalamin (V), which comprises the steps of methylating cyanocobalamin (I) or hydroxocobalamin (II) in the presence of a cyan ion-trapping agent, a reducing agent (III) and a water-soluble methylating agent (IV) in an aqueous solution or a hydrous organic solvent; and then precipitating the reaction product as crystals or precipitates.
In the present invention, in the case that a cyan ion-trapping agent is used, examples of the cyan ion-trapping agent include metals or metal salts such as ferrous sulfate, iron powder, Mohr""s salt, ferrous chloride, cobalt chloride, nickel chloride and zinc chloride, and particularly preferred are ferrous sulfate and/or cobalt chloride. These metals or metal salts may be used solely or in combination.
The cyan ion-trapping agent may be used in a small amount, and the amount is usually from 1 to 30% by weight and more preferably from 1 to 10% by weight to cyanocobalamin (I) or hydroxocobalamin (II).
Finally, the use of a reaction solvent is not particularly limited, and in the case of using a solvent, it is not particularly limited as far as it is inert to cyanocobalamin (I), hydroxocobalamin (II), trimethylsulfur derivative (VI) or methylcobalamin (V). The reaction solvent is usually an aqueous solution or a hydrous organic solvent. As the organic solvent, preferred is usually a water-soluble one, and examples thereof include lower alcohols such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol, sec-butanol and t-butanol; various esters such as methyl formate, ethyl formate, methyl acetate, ethyl acetate and isopropyl acetate; various ketones such as acetone, 2-butanone and 3-methyl-2-butanone; cyclic ethers such as THF and dioxane; acetonitrile, DMF, DMSO, pyridine etc.; and mixtures containing one or more of them.
The reaction temperature in the present invention is also not particularly limited, but the reaction is conducted at a temperature of usually 0 to 90xc2x0 C., preferably 10 to 70xc2x0 C. and more preferably 15 to 50xc2x0 C.
A more preferred result is obtained by conducting the reaction under a stream of an inert gas such as nitrogen and/or in the dark place (under infrared ray).
In order to explain the present invention specifically, Examples will be described in the following, but the invention is by no means limited thereto.